Signaling through the mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinases (PI3Ks)/AKT pathway is triggered by extracellular stimulation and regulates a variety of biological processes, such as proliferation, differentiation and cell death. Both pathways are often activated in many cancers by mutations or overexpression of upstream molecules. These pathways interact with each other to regulate tumor growth and, thus, they are potential targets in treating cancer.
Phosphatidylinositol 3-kinases (PI3Ks) comprise a family of lipid kinases that catalyze the transfer of phosphate to the D-3′ position of inositol lipids to produce phosphoinositol-3-phosphate (PIP), phosphoinositol-3,4-diphosphate (PIP2) and phosphoinositol-3,4,5-triphosphate (PIP3) that, in turn, act as second messengers in signaling cascades by docking proteins containing pleckstrin-homology, FYVE, Phox and other phospholipid-binding domains into a variety of signaling complexes often at the plasma membrane (Vanhaesebroeck et al., Annu. Rev. Biochem 70:535 (2001); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615 (2001)). Of the two Class 1 PI3Ks, Class 1A PI3Ks are heterodimers composed of a catalytic p110 subunit (α, β, δ isoforms) constitutively associated with a regulatory subunit that can be p85α, p55α, p50α, p85β or p55γ. The Class 1B sub-class has one family member, a heterodimer composed of a catalytic p110γ subunit associated with one of two regulatory subunits, p101 or p84 (Fruman et al., Annu Rev. Biochem. 67:481 (1998); Suire et al., Curr. Biol. 15:566 (2005)). The modular domains of the p85/55/50 subunits include Src Homology (SH2) domains that bind phosphotyrosine residues in a specific sequence context on activated receptor and cytoplasmic tyrosine kinases, resulting in activation and localization of Class 1A PI3Ks. Class 1B PI3K is activated directly by G protein-coupled receptors that bind a diverse repertoire of peptide and non-peptide ligands (Stephens et al., Cell 89:105 (1997)); Katso et al., Annu. Rev. Cell Dev. Biol. 17:615-675 (2001)). Consequently, the resultant phospholipid products of class I PI3K link upstream receptors with downstream cellular activities including proliferation, survival, chemotaxis, cellular trafficking, motility, metabolism, inflammatory and allergic responses, transcription and translation (Cantley et al., Cell 64:281 (1991); Escobedo and Williams, Nature 335:85 (1988); Fantl et al., Cell 69:413 (1992)).
PIP2 and PIP3 frequently recruit Akt, the product of the human homologue of the viral oncogene v-Akt, to the plasma membrane where it acts as a nodal point for many intracellular signaling pathways important for growth and survival (Fantl et al., Cell 69:413-423(1992); Bader et al., Nature Rev. Cancer 5:921 (2005); Vivanco and Sawyer, Nature Rev. Cancer 2:489 (2002)). Aberrant regulation of PI3K, which often increases survival through Akt activation, is one of the most prevalent events in human cancer and has been shown to occur at multiple levels. The tumor suppressor gene PTEN, which dephosphorylates phosphoinositides at the 3′ position of the inositol ring and in so doing antagonizes PI3K activity, is functionally deleted in a variety of tumors. In other tumors, the genes for the p110α isoform, PIK3CA, and for Akt are amplified and increased protein expression of their gene products has been demonstrated in several human cancers. Furthermore, mutations and translocation of p85α that serve to up-regulate the p85-p110 complex have been described in human cancers. Finally, somatic missense mutations in PIK3CA that activate downstream signaling pathways have been described at significant frequencies in a wide diversity of human cancers (Kang at el., Proc. Natl. Acad. Sci. USA 102:802 (2005); Samuels et al., Science 304:554 (2004); Samuels et al., Cancer Cell 7:561-573 (2005)). These observations show that deregulation of phosphoinositol-3 kinase and the upstream and downstream components of this signaling pathway is one of the most common deregulations associated with human cancers and proliferative diseases (Parsons et al., Nature 436:792 (2005); Hennessey at el., Nature Rev. Drug Disc. 4:988-1004 (2005)).
Further, over-activation of mitogen-activated protein (MAP) kinase cascade is known to play an important role in cell proliferation and differentiation. This pathway can be activated when a growth factor binds to its receptor tyrosine kinase. This interaction promotes RAS association with RAF and initiates a phosphorylation cascade through mitogen activated protein kinase (MEK) to ERK. Phosphorylation of MEK appears to increase its affinity and its catalytic activity toward ERK as well as is affinity for ATP.
The MAP kinase pathway is deregulated, often through mutations that result in ectopic protein activation, in roughly ⅓ of human cancers. This deregulation in turn results in a wide array of cellular changes that are integral to the etiology and maintenance of a cancerous phenotype including, but not limited to, the promotion of proliferation and evasion of apoptosis (Dhillon et al., Oncogene, 2007, 26: 3279-3290). Inhibition of this pathway is known to be beneficial in proliferative diseases. MEK is an attractive therapeutic target because the only known substrates for MEK phosphorylation are the MAP kinases, ERK1 and ERK2. MEK is frequently activated in tumors that have mutations in the RAS or RAF oncogenes. Constitutive activation of MEK/ERK has been found in pancreatic, colon, lung, kidney and ovarian primary tumor samples.
Inhibition of MEK has been shown to have potential therapeutic benefit in various diseases in several studies such as: (a) Tumor and Leukemia: Evidence of Efficacy in Tumor Models (Nature-Medicine 5(7): 810-816, 1999; Tracet et al, AACR Apr. 6-10, 2002, Poster #5426; Tecle, H. IBC 2nd International Conference of Protein Kinases, Sep. 9-10, 2002, J. Clin. Invest. 108(6), 851-859, 2001), (b) Pain: Evidence of Efficacy in Pain Models (J. Neurosci. 22:478, 2002; Acta Pharmacol Sin. 26:789 2005; Expert Opin Ther Targets. 9:699, 2005; Mol. Pain. 2:2, 2006), (c) Stroke: Evidence of Efficacy in Stroke Models Significant Neuroprotection against Ischemic Brain Injury by Inhibition of the MEK (J. Pharmacol. Exp. Ther. 304:172, 2003; Brain Res. 996:55, 2004), (d) Diabetes: Evidence In Diabetic Complications. (Am. J. Physiol. Renal. 286, F120 2004), (e) Inflammation: Evidence of Efficacy in Inflammation Models. (Biochem Biophy. Res. Com. 268:647, 2000), and (f) Arthritis: Evidence of efficacy in experimental osteoarthritis. (Arthritis & (J. Clin. Invest. 116:163. 2006).
The PI3K pathway interacts extensively with the MAPK pathway. These pathways share common upstream activators, and they are both activated by oncogenic RAS and appear to provide some compensatory signaling when one or the other is inhibited.
In spite of numerous treatment options for patients with cancer, there remains a need for effective and safe therapeutic agents and a need for new combination therapies that can be administered for the effective long-term treatment of cancer. It has been surprisingly discovered that the combination of an effective amount of the p110α-specific phosphatidylinositol 3-kinase (PI3K) inhibitor compound (S)-pyrrolidine-1,2-dicarboxylic acid 2-amide 1-(4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl)-amide with an effective amount of at least one MEK inhibitor compound of the present invention, in particular 6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethoxy)-amide or (S)-5-fluoro-2-(2-fluoro-4-(methylthio)phenylamino)-N-(2-hydroxypropoxy)-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide, results in unexpected improvement in the treatment of proliferative diseases, particularly cancer. When administered simultaneously, sequentially or separately, this specific phosphatidylinositol 3-kinase (PI3K) inhibitor compound and the MEK inhibitor compound of the present invention interact in a synergistic manner to strongly inhibit cell proliferation. This unexpected synergistic reaction allows reduction in the dose required for each compound, leading to a reduction in the side effects and enhancement of the long-term clinical effectively of the compounds in treatment.